The present invention relates to microscopes, and more particularly, to methods and apparatus for viewing and manipulating cells inside living tissue with a microscope.
Before tissue can be viewed using conventional microscopes, it must usually be removed from its host organism, especially when features below the surface of the tissue are viewed. However, living tissue cannot survive long after removal from its host without sophisticated support equipment. Further, the tissue may change if the support equipment does not precisely duplicate its natural environment. Although a few conventional microscopes (e.g., surgical microscopes) have been designed to view living tissue without removing it from its host, these microscopes have limited resolution. Therefore, small features cannot be seen with these microscopes.
Due to the inherent limitations of conventional microscopes, many features of living tissue have not been viewed directly. For instance, physical changes in the human brain resulting from internal processes have not been viewed at the cellular level. As a result, information such as how quickly connections (e.g., synapse connections) are made and lost within the brain is unknown. Further, viewing removed brain tissue does not permit clear understanding of these processes because complex behavior (e.g., speech or learning) cannot be studied when the tissue is removed from its host. The inability of conventional microscopes to view brain cell connections is particularly frustrating because it is envisioned that viewing these connections could answer questions concerning the causes of brain dysfunction such as Alzheimer""s disease.
One of the reasons tissue must be removed from its host before it may be viewed by most conventional microscopes is that the tissue must be highly illuminated to be seen through the microscopes. Confocal optical microscopes eliminate this problem by illuminating the tissue with a laser aimed at the tissue through the lens of the microscope. These microscopes make it possible to view an object without an external illumination source. However, conventional confocal optical microscopes cannot view more than a very short distance (i.e., about 200 nm) below the surface of the tissue. Thus, deeper tissue cannot be viewed without separating the tissue from the living organism.
Conventional microscopes and methods of use have other disadvantages which limit their usefulness when viewing tissue inside a host. For instance, stains are ordinarily applied to tissue before being viewed with microscopes to improve the optical attributes of features within the tissue. However, the amount of stain used to produce suitable optical attributes frequently kills or injures cells in the tissue and sometimes harms the host. Therefore, conventional methods of applying stain are generally not appropriate when examining living tissue inside a host organism.
Among the several objects and features of the present invention may be noted the provision of a microscope attachment capable of viewing internal features of tissue without removing the tissue from its host; the provision of a microscope which enables small amounts of stains and/or other fluids to be precisely directed toward a particular site in tissue within the field of view of the microscope; the provision of a microscope capable of precisely positioning instruments for manipulation of tissue within the field of view of the microscope; and the provision of a microscope having a fluid delivery system which delivers fluid to a site within the field of view of the microscope in amounts which are effective and substantially nontoxic.
Briefly, apparatus of this invention is an assembly for viewing cells inside tissue of a living organism. The assembly includes a confocal microscope having an objective for magnifying an image positioned at a focal plane of the objective and a light source adapted to direct light through the objective. The assembly also includes a rigid elongate tube extending from the objective to a tip sized and shaped for penetrating the tissue of the living organism. The tube has a hollow interior aligned with the objective. The interior of the tube is free of fiberoptic bundles and cover glasses. The assembly also includes a unitary cylindrical lens positioned in the hollow interior of the tube for transmitting light from the light source to the cells inside the tissue of the living organism adjacent the tip to illuminate the cells. The lens has a focal plane adjacent the tip positioned at a location corresponding to the illuminated cells, an image plane opposite the tip positioned at the focal plane of the microscope objective and a sufficient resolution for transmitting an image of the illuminated cells positioned at the focal plane of the lens to the focal plane of the objective.
In another aspect, the invention includes an attachment for use with a microscope. The attachment includes a rigid elongate tube having a hollow interior extending to a tip having a width of less than about three millimeters to permit the tip to be inserted into tissue of a living organism. The attachment further comprises a unitary cylindrical lens positioned in the hollow interior of the tube for transmitting an image of a specimen positioned at a focal plane of the lens adjacent a front end thereof to an image plane of the lens adjacent a rear end thereof opposite said front end. The lens is free of fiberoptic bundles and cover glasses between the focal plane of the lens and the image plane of the lens. The lens has a sufficient resolution to permit cells of the tissue to be viewed through the lens with a microscope. The attachment also has a mount for mounting the tube and lens on a microscope in a position wherein the image plane of the lens corresponds with a focal plane of the microscope objective.
In yet another aspect, the invention includes a method of viewing cells inside tissue of living organisms. The method comprises the steps of mounting a cylindrical lens adjacent an objective of a microscope and adjusting the lens so an image plane of the lens corresponds with a focal plane of the microscope objective. The lens and the objective are simultaneously moved as a unit to penetrate the tissue and position the lens so the cells lie within a focal plane of the lens.
Other objects and features of the present invention will be in part apparent and in part pointed out hereinafter.